The World Health Organization estimates that one in three human beings is believed to be infected with Mycobacterium tuberculosis (Styblo, K., Reviews of Infectious Diseases. Vol. II, Suppl. 2, March-April, 1989; Bloom and Murray, Science 257:1055-1067, 1992). Over the past decade, there has been a recent resurgence in the incidence of tuberculosis in developed countries that has coincided with the AIDS epidemic (Snider and Roper, N. England J. Med. 326:703-705 (1992)). Because of their impact as major human pathogens and as a result of their profound immunostimulatry properties, mycobacteria have long been intensively studied. In the early 1900s, an attenuated mycobacterium, Mycobacerium(M.) bovis Bacille Calmette-Guerin (M. bovis BCG or BCG), was isolated for use as a vaccine against tuberculosis (Calmette et al. Acad. Natl. Med. (Paris), 91:787-796, 1924; reviewed in Collins, F. M., Bacterial Vaccines (R. Germanier, ed.), Academic Press, pp. 373-418, 1984). Although the efficacy of this vaccine against tuberculosis varied considerably in different trials, and the reasons for its variable efficacy have yet to be resolved, BCG is among the most widely used human vaccines (Luelmo, F., Am. Rev. Respir. Dis. 125:70-72, 1982; Fine, P. E. M., Reviews of Infectious Diseases II (supp. 2), 5353-5359, 1989).
The recent application of molecular biological technology to the study of mycobacteria has led to the identification of many of the major antigens that are targets of the immune response to infection by mycobacteria (Kaufmann, S. H. E., Immunol. Today 11:129-136, 1990; Young, R. A., Ann. Rev. Immunol. 8:401-420, 1990; Young et al., Academic Press Ltd., London, pp. 1-35, 1990; Young et al., Mol. Microbiol. 6:133-145, 1992)) and to an improved understanding of the molecular mechanisms involved in resistance to antimycobacterial antibiotics (Zhang et al., Nature 358:591-593, 1992; Telenti et al., Lancet 341:647-650, 1993). The development of tools that permit molecular genetic manipulation of mycobacteria has also allowed the construction of recombinant BCG vaccine vehicles (Snapper et al., Proc. Natl. Acad. Sci. USA 85:6987-6991, 1988; Husson et al., J. Bacteriol. 172:519-524, 1990; Martin et al., B. Nature 345:739-743, 1990; Snapper et al., Mol. Microbiol. 4:1911-1919, 1990; Aldovini and Young, Nature 351:479-482, 1991; Jacobs et al., Methods Enzymol. 204:537-555, 1991; Lee et al., Proc. Natl. Acad. Sci. USA 88:3111-3115, 1991; Stover et al., Nature 351: 456-460, 1991; Winter et al., Gene 109:47-54, 1991; Donnelly-Wu et al., Mol. Microbiol. 7:407-417, 1993)). Genome mapping and sequencing projects are providing valuable information about the M. tuberculosis and M. leprae genomes that will facilitate further study of the biology of these pathogens (Eiglmeier et al., Mol. Microbiol., in press, 1993; Young and Cole, J. Bacteriol. 175:1-6, 1993).
Despite these advances, there are two serious limitations to our ability to manipulate these organisms genetically. First, very few mycobacterial genes that can be used as genetic markers have been isolated (Donnelly-Wu et al., Mol. Microbiol. 7:407-417, 1993)). In addition, investigators have failed to obtain homologous recombination in slow growing mycobacteria, such as M. tuberculosis and M. bovis BCG (Kalpana et al., Proc. Natl. Acad. Sci. USA 88:5433-5447, 1991; Young and Cole, J. Bacteriol. 175:1-6, 1993)), although homologous recombination has been accomplished in the fast growing Mycobacterium smegmatis (Husson et al., J. Bacteriol. 172: 519-524, 1990)).